1. Field of the Invention
This invention relates to mutant proteolytic enzymes having improved properties relative to the wild-type enzyme, to genetic constructs which code for the mutant proteolytic enzymes, to methods of predicting mutations which enhance the stability of the enzyme, and to methods of producing the mutant proteolytic enzymes.
2. Description of the Related Art
Subtilisins are a family of extracellular proteins having molecular weights in the range of 25,000-35,000 daltons and are produced by various Bacillus species. These proteins function as peptide hydrolases in that they catalyze the hydrolysis of peptide linkages in protein substrates at neutral and alkaline pH values. Subtilisins are termed serine proteases because they contain a specific serine residue which participates in the catalytic hydrolysis of peptide substrates. A subtilisin enzyme isolated from soil samples and produced by Bacillus lentus for use in detergent formulations having increased protease and oxidative stability over commercially available enzymes under conditions of pH 7 to 10 and at temperature of 10 to 60.degree. C. in aqueous solutions has been disclosed in copending patent application Ser. No. 07/398,854, filed on Aug. 25, 1989. This B. lentus alkaline protease enzyme (BLAP, vide infra) is obtained in commercial quantities by cultivating a Bacillus licheniformis ATCC 53926 strain which had been transformed by an expression plasmid which contained the wild type BLAP gene and the B. licheniformis ATCC 53926 alkaline protease gene promoter.
Industrial processes generally are performed under physical conditions which require highly stable enzymes. Enzymes may be inactivated by high temperatures, pH extremes, oxidation, and surfactants. Even though Bacillus subtilisin proteases are currently used in many industrial applications, including detergent formulations, stability improvements are still needed. Market trends are toward more concentrated detergent powders, and an increase in liquid formulations. Increased shelf stability and oxidative stability, with retention of catalytic efficiency are needed. It is therefore desirable to isolate novel enzymes with increased stability, or to improve the stability of existing enzymes, including subtilisin proteases such as BLAP.
The stability of a protein is a function of its three dimensional structure. A protein folds into a three dimensional conformation based upon the primary amino acid sequence, and upon its surrounding environment. The function and stability of a protein are a direct result of its three dimensional structure.
A large body of information has been published which describes changes in enzyme properties as a result of alterations in the primary amino acid sequence of the enzyme. These alterations can result from random or site specific alterations of the gene which expresses the enzyme using genetic engineering techniques. Random approaches mutagenize total cellular DNA, followed by selection for the synthesis of an enzyme with improved properties. This approach requires neither knowledge of the three dimensional structure of the enzyme, nor any predictive capability on the part of the researcher. Site directed mutagenesis, on the other hand, requires a rational approach for the introduction of amino acid changes. In this approach one or more amino acids may be replaced by other residues by altering the DNA sequence which encodes the protein. This can be accomplished using oligonucleotide directed in vitro mutagenesis. The following references teach site-directed mutagenesis procedures used to generate specific amino acid substitution(s): Hines, J.C., and Ray, D.S. (1980) Gene 11:207-218; Zoller, M.J., and Smith, M. (1982) Nucleic Acids Res. 10:6487-6500; Norrander, J., et al. (1983) Gene 26:101-106; Morinaga, Y., et al. (1984) Bio/Technology 2:636-639; Kramer, W., et al. (1984) Nucleic Acids Res. 12:9441-9456; Carter, P., et al. (1985) Nucleic Acids Res. 13:4431-4443; Kunkel, T.A. (1985) Proc. Natl. Acad. Scio USA 82:488-492; Bryan, P., et al. (1986) Proc. Natl. Acad. Sci. USA 83:3743-3745.
A rational approach may or may not require knowledge of a protein's structure. For example, patent application WO 89/06279 describes the comparison of the primary amino acid sequence of different subtilisins while contrasting differences in physical and chemical properties. The primary amino acid sequences of the different subtilisins are aligned for the greatest homology, while taking into account amino acid insertions, deletions, and total number of amino acids.
Currently, the amino acid sequences of at least 10 subtilisin proteases have been published. Eight of these subtilisins were isolated from species of Bacilli, and include subtilisin 168 (Stahl, M.L., and Ferrari, E. (1984) J. Bacteriol. 158:411-418), subtilisin BPN' (Vasantha, N., et al., (1984) J. Bacteriol. 159:811-819), subtilisin Carlsberg (Jacobs, M., et al. (1985) Nucleic Acids Res. 13:8913-8926), subtilisin DY (Nedkov, P., et al. (1985) Biol. Chem. Hoppe-Seyler 366:421-430), subtilisin amylosacchariticus (Kurihara, M., et al. (1972) J. Biol. Chem. 247:5619-5631), subtilisin mesenticopeptidase (Svendsen, I., et al. (1986) FEBS Lett. 196:228-232), subtilisin 147 and subtilisin 309 (Hastrup et al. (1989) WO 9/06279), subtilisin PB92 (Van Eekelen et al. (1989) EP 328229), and subtilisin BLAP (Ladin, B., et al. (1990) Society for Industrial Microbiology Annual Meeting, Abstract P60). The remaining two subtilisin sequences are thermitase from the fungus Thermoactinomyces vulgaris (Meloun, B., et al. (1985) FEBS Lett. 183:195-200), and proteinase K from the fungus Tritirachium album limber (Jany, K.-D., and Mayer, B. (1985) Biol. Chem. Hoppe-Seyler 366:485-492).
Methods for obtaining optimum alignment of homologous proteins are described in Atlas of Protein Sequence and Structure, Vol. 5, Supplement 2 (1976) (Dayhoff, M.O., ed., Natl. Biomed. Res. Found., Silver Springs, Md.). This comparison is then used to identify specific amino acid alterations which might produce desirable improvements in the target enzyme. Wells, J.A., et al. (1987) Proc. Natl. Acad. Sci. USA 84:1219-1223, used primary sequence alignment to predict site directed mutations which affect the substrate specificity of a subtilisin. Using the alignment approach WO 89/06279 teaches the construction of mutant subtilisins having improved properties including an increased resistance to oxidation, increased proteolytic activity, and improved washing performance for laundry detergent applications. Patent applications WO 89/09819, and WO 89/09830 teach improvement in the thermal stability of subtilisin BPN' by the introduction of one or more amino acid changes based on the alignment of the primary amino acid sequences of subtilisin BPN' with the more thermal stable subtilisin Carlsberg. From hereon, amino acids will be referred to by the one or three letter code as defined in Table 1.